The use of proteases in heavy duty liquid detergent formulations is complicated by their limited stability in solution. Two processes which limit the shelf-life of a protease in an aqueous liquid detergent are denaturation and autolysis (self-digestion). Considerable efforts have been devoted to the stabilization of enzymes in aqueous liquid detergent compositions, which represent a medium that is problematical for the preservation of enzyme activity during storage and distribution.
Denaturation of proteases may be minimized by selection of optimal formulation components such as actives, builders, etc., and conditions such as pH, so that acceptable enzyme stability is achieved. Self-digestion of proteases may be minimized by inclusion of a protease inhibitor. The inhibitor is released from the enzyme upon dilution in the wash and the proteolytic activity is restored.
Various protease inhibitors are known in the art. For example, U.S. Pat. No. 4,261,868 (Unilever) teaches the use of borax as a protease inhibitor and both U.S. Pat. No. 4,243,546 (Drackett) and GB-A-1 354 761 (Henkel) teach the use of carboxylic acids as protease inhibitors. Various combinations of these protease inhibitors are also known in the art. U.S. Pat. No. 4,305,837 (Procter & Gamble), for example, teaches the combination of carboxylic acids and simple alcohols and U.S. Pat. No. 4,404,115 (Unilever) teaches the combination of borax and polyols as protease inhibitors. U.S. Pat. No. 4,537,707 (Procter & Gamble) teaches the combination of borax and carboxylates as protease inhibitors.
It is also known to use mutant subtilisin proteases which have been modified by substitution at an amino acid site. U.S. Pat. No. 4,760,025 (Genencor), for example, claims subtilisin mutants with amino acid substitutions at amino acid sites 32, 155, 104, 222, 166, 64, 33, 169, 189, 217 or 157 which are different from subtilisins naturally produced by B. amyloliquefaciens. A mutant protease whereby methionine at position 222 has been replaced by alanine, is shown to have an improved oxidation stability in the presence of bleach.
WO-A-89/06279 (Novo/Nordisk) discloses subtilisin mutants having modified chemical characteristics. In particular it is shown that a subtilisin mutant which has been modified at positions 195 and/or 222 exhibit an enhanced oxidation stability in the presence of peracetic acid. In a publication from Novo/Nordisk in "Biopapers Journal" Vol. 10, Issue 5 november/december 1990, page 11-14, it is disclosed that the commercially available protease Durazym is an engineered Savinase protease made by changing glycine 195 to glutamic acid and methionine 222 to alanine in the protease.
We have now surprisingly found that the mutant subtilisin enzymes which have been modified at positions 195 and 222 are of exceptional value for formulating stable, liquid detergent compositions. First, they are remarkably stable in the absence of any bleaching agent, and secondly, they are remarkably compatible with any other enzymes present in the composition, such as lipase or amylase.
WO-A-87/04461 (Amgen) discloses the substitution in Bacillus subtilisins of alternative amino acids (i.e. serine, valine, threonine, cysteine, glutamine and isoleucine) for ASN, GLY or ASN-GLY sequences (specifically at position 218). These mutations are said to increase the stability of the enzyme at high temperatures or over a broader pH range than the wild type enzyme. WO-A-88/08033 (Amgen) claims mutations which modify calcium-binding capacity (to replace an amino acid with a negatively charged residue such as ASP or GLU) and optionally a deletion and/or replacement of either residue of ASN-GLY sequences which results in better pH and thermal stability and higher specific activities. The reference claims that sites 41, 75, 76, 77, 78, 79, 80, 81, 208, and 214 may be replaced by a negatively charged amino acid and ASN may be replaced by SER, VAL, THR, CYS, GLU, or ILE in ASN-GLY sequences.
These references do not disclose detergent compositions comprising the subtilisin mutants of the subject invention or the advantages provided by the use of these mutants in these detergent compositions.
WO-A-89/06279 (Novo/Nordisk) discloses the subtilisin mutants which are used in the liquid detergent compositions of the present invention. Although the use of such mutants in bleach containing washing preparations is disclosed (Table VI), there is no teaching of the use of these mutants in detergent composition which do not contain any bleaching agents. To the contrary, the skilled man would not be inclined to make use of such mutants applications where oxidation stability does not seem to offer any advantages, because in general the proteolytic activity of the mutants is lower than that of the native enzyme. Consequently, there is no disclosure of the use of these mutants in specific detergent compositions and no teaching or disclosure that the mutant enzymes will have enhanced stability in these specifically defined compositions.
Furthermore, it is known that lipase has a tendency to be less stable in the presence of protease than in the absence of protease; surprisingly, it now was found that the mutant subtilisin enzymes of the present invention are remarkably more compatible with lipase enzyme than wild-type subtilisin enzyme.
Finally, it was found that the mutant subtilisin enzymes of the present invention are remarkably more compatible with amylase enzyme than wild-type subtilisin enzyme.